Pharmacological methods

Data handling and pharmacological parameters: Data presented as mean ± SEM or SE or with 95% confidence limits (cl) unless otherwise indicated. Agonist potencies are given in percent or are expressed as pEC50 values (negative decadic logarithm of the molar concentration of the agonist producing 50% of the maximal response) and were corrected according to the long term mean value of the reference agonist histamine in our laboratory

(guinea pig atrium (H2): pEC50 = 6.00 for histamine; guinea pig ileum (H1): pEC50 = 6.70 for histamine). Maximal responses are expressed as Emax values (percentage of the maximal response to a reference compound). Antagonist affinities are expressed as either an apparent pA2 or a full pA2 value. The apparent pA2 value was calculated from the following equation:

pA2 = −log c(B) + log(r − 1), where c(B) is the molar concentration of antagonist and r the ratio of agonist EC50 measured in the presence and absence of antagonist.⁴⁵ The full pA2 value was determined according to the method of Arunlakshana and Schild⁴⁶ using antagonist concentrations over 1-2 log units. Noncompetitive antagonists are characterized by estimation of a pD'2 value according to the equation: pD'2 = −log c(B) + log(100/Emax − 1).⁴⁷ Where appropriate, differences between means were determined by Student's t-test, after checking the homogeneity of the variances; P values < 0.05 were considered to indicate a significant difference between the mean values being compared.

Histamine H1 receptor assay on the isolated guinea pig ileum: Guinea pigs of either sex (250-500 g) were stunned by a blow on the neck and exsanguinated. The ileum was rapidly removed, rinsed and cut into segments of 1.5-2 cm length. The tissues were mounted isotonically (preload of 5 mN) in a jacketed 20-mL organ bath that was filled with Tyrode's solution of the following composition [mM]: NaCl 137, KCl 2.7, CaCl2 1.8, MgCl2 1.0, NaH2PO4 0.4, NaHCO3 11.9, and glucose 5.0. The solution additionally contained atropine to block cholinergic M receptors at a concentration not affecting H1 receptors (0.1 μM). The solution was aerated with 95% O2-5% CO2 and warmed to a constant temperature of 37 °C.

During an equilibration period of 80 min, the tissues were stimulated three times with histamine (1 μM, then 10 μM) followed by washout. Antagonists: Up to four cumulative concentration-response curves were determined on each tissue: a first to histamine (0.01-30 μM), and the 2^nd – 4^th to histamine in the presence of increasing concentrations of antagonist (incubation time 10–15 min). pEC50 differences were not corrected since four successive curves for histamine were superimposable (n > 10).

Histamine H2 receptor assay on isolated guinea pig right atrium (spontaneously beating): Hearts were rapidly removed from guinea pigs used for studies on the ileum (see

solution under a diastolic resting force of 5 mN in a jacketed 20 mL-organ bath of 32.5 °C as previously described.⁶ The bath fluid (composition [mM]: NaCl 118.1, KCl 4.7, CaCl2 1.8, MgSO4 1.64, KH2PO4 1.2, NaHCO3 25.0, glucose 5.0, sodium pyruvate 2.0) was gassed with 95% O2-5% CO2 and additionally contained (RS)-propranolol (0.3 μM) and mepyramine (1 μM). Experiments were started after 30 min of continuous washing and an additional equilibration period of 15 min. Antagonists: Two successive concentration-frequency curves to histamine (0.1-30 μM) were established, the first in the absence and the second in the presence of the compound under study (incubation time 30 min). pEC50 differences were not corrected since two successive curves for histamine were virtually superimposable (n > 10).

Agonists: Two successive concentration-frequency curves were established, the first to histamine (0.1-30 μM) and the second for the agonist under study in the absence or presence of cimetidine (30 μM, 30 min incubation time). Furthermore, the sensitivity to 30 μM cimetidine was routinely checked at the end of each H2 agonist concentration-effect curve established in the absence of an H2 receptor antagonist, and a significant reduction of frequency was always observed after 15–30 min.

Calcium assays with U-373 MG cells

Cells and culture conditions: The human U-373 MG (HTB 17) glioblastoma/astrocytoma cell line was obtained from the American Type Culture Collection (Rockville, MD, USA).

Cell banking and quality control were performed according to the "seed stock concept". Cells were grown in Eagle´s minimum essential medium containing L-glutamine, 2.2 g/l NaHCO3, 110 mg/l sodium pyruvate (Sigma, Deisenhofen, FRG), and 10% FCS (GIBCO, Eggenstein, FRG The cells were cultured in a water-saturated atmosphere of 95% air and 5% carbon dioxide at 37 °C in 75-cm² culture flasks (Costar, Tecnomara, Fernwald, FRG) and were serially passaged following trypsinisation using trypsin (0.05%) / EDTA (0.02%) (Roche Diagnostics, Mannheim, GER).

Preparation of the cells: Adherently growing U-373 MG cells were trypsinized and transferred from a 75-cm² flask to 175-cm² flask (Nunclon, 178883, Nunc, Wiesbaden, and Germany) 5-6 days before the experiment. At approx. 80% confluence cells were trypsinized, the suspension (2-4·10⁵ cells per ml), was centrifuged for 10 min at 200 g and room

temperature. After resuspension in 10 ml of loading buffer (25 mM HEPES (Sigma, Deisenhofen, GER), 120 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose), pH 7.4, cell number was determined with a hemocytometer (Neubauer, improved), and the cells were adjusted to a density of 1.3·10⁶ per ml by addition of an appropriate volume of loading buffer.

Loading of the cells with Ca²⁺ indicator Fura-2/AM: To three volumes of the prepared cell suspension, one volume of loading dispersion was added, before the cells were incubated in the dark at room temperature for 30 min. The loading dispersion was freshly made by mixing 10 ml of loading buffer, containing 2 % bovine serum albumin (BSA), with 50 μl of Pluronic-F-127 (Molecular Probes, Eugene, Oregon, USA) (20 % in DMSO), and 40 μl of Fura-2/AM (Molecular Probes, Eugene, Oregon, USA) (1 mM in anhydrous DMSO).

Cells were centrifuged (200 g, 7 min), resuspended in fresh loading buffer and allowed to stand for another 30 min at room temperature in the dark. After two washing/centrifugation cycles (loading buffer, 200 g, 7 min) and adjustment of the cell number to a value of 10⁶ /ml, cells were incubated for at least 15 min at 20 °C in the dark.

Fluorimetric determination of intracellular [Ca²⁺]: 1 ml-aliquots of loading buffer were filled into disposable acrylic cuvettes (Sarstedt, No. 67.755, Nuembrecht, GER), which were thermostatted at 30 ° C in an incubator hood (Infors AG, Bottmingen, CH). Immediately after addition of 1 ml of the Fura-2/AM loaded cell suspension and a magnetic stirrer, the cuvette was placed into the thermostatted (25 °C) stirred cell holder of a LS 50 B Luminescence Spectrometer (Perkin Elmer, Ueberlingen, GER), equipped with a fast filter accessory.

Fluorescence signals were registered (instrument settings: excitation 340/380 nm, emission 510 nm, slits 10 nm, resolution 0.1, stirrer low) for 300 s after addition of 10 μl of 6 mM histamine dihydrochloride dissolved in millipore water.

Calculation of Ca²⁺ concentrations: Calcium concentrations were calculated from dual wavelength fluorescence intensities according to the Grynkiewicz equation:

[ ]

^{(R−R )}

were Kd (224 nM) is the dissociation constant of the Fura-2-Ca²⁺-complex, R is the experimental fluorescence ratio value (F340 / F380), Rmin and Rmax are the fluorescence value ratios (F340 / F380) under Ca²⁺-free and Ca²⁺-saturation conditions, respectively, and SFB is the ratio of fluorescence intensities for Ca²⁺-free/Ca²⁺-bound indicator, measured at 380 nm. Rmin, Rmax and SFB were determined by calibration experiments, performed in every test series.

To measure Rmax, 10 μl of an aqueous solution of 2 % digitonin (Sigma, Deisenhofen, GER), were pipetted into the cuvette, wheras Rmin was determined after subsequent addition of 50 μl of a 0.6 M EGTA solution (in 1 M Tris/HCl, pH 8.7).

Determination of histamine H1 receptor antagonists on U-373 MG cells:⁴⁸

To determine the histamine H1 receptor antagonism on U-373 MG cells the inhibition of the (submaximal) increase in [Ca²⁺]i elicited by 30 μM histamine was measured. Prior to stimulation with histamine the cells were incubated with the putative antagonists for 15 min in the dark with stirring. The inhibition (%) was calculated as follows:

Inhibition [%] = ( 100 - ) · 100

[Ca ²⁺ ] i -increase (control) [µM]

[Ca ²⁺ ] _i -increase (antagonist + 30 µM hist.) [µM]

At least seven concentrations of the antagonists were used and at least two independent experiments were carried out on different days. The mean values of the resulting inhibition curves were used to calculate IC50 and pKB values, respectively.

Calculation of IC50 values: IC50- values were calculated from at least two antagonist concentrations [B], inhibiting the agonist-stimulated increase in intracellular [Ca²⁺] between 20 and 80 %. The mean percentual inhibition values P with SEM < 10 %, determined from at least 3 independent experiments, performed on different days, were logit transformed, according to the equation

logit (P) = log P

100 - P

and IC50 values (logit P = 0) were determined from the plot logit (P) versus log [B] with the slope n according to

log P

100 - P = n log [B] - n log IC⋅ ⋅ 50 by linear regression with Fig. P (Biosoft, Cambridge, UK).

Steady-sstate GTPase activity assay and receptor ligand binding assay on guinea pig and human histamine H2 receptor G_sαS fusion proteins: These investigations were performed according to the procedure described by Kelley et al.²⁴

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Synthesis and Pharmacological Activity of N-Acyl-N’-[3-(2-amino-4-methylthiazol-5-yl)propyl]guanidines: Towards Improved H

2

Receptor Selectivity

Only such substances can be anchored at any particular part of the organism, as fit into the molecules of the recipient complex like a piece of mosaic finds its place in the pattern.

Paul Ehrlich, 1956 4.1. Introduction

The clinical use of compounds active on the histamine H2 receptor (H2R) is mainly restricted to antagonists, which are useful in the treatment of peptic ulcers. However, some efforts have been made with histamine H2R agonists in the treatment of patients suffering from congestive heart failure.^1,2 Histamine H2 receptor agonists can be divided in two structural classes of compounds:

histamine analogues and dimaprit^3,4 (containing amine), and impromidine analogues (containing guanidine)^5,6 including arpromidine and related imidazolylpropylguanidines⁷. Histamine and dimaprit both show moderate histamine H2R agonistic activity at the guinea pig right atrium, however, the H1-receptor agonistic potency of dimaprit on the isolated guinea pig ileum (H1) proved to be less than 0.0001% of that of histamine.⁸

Analogues of impromidine as described by Sterk⁵ and Buschauer⁷ are potent histamine H2R agonists, and some of them show considerable H1- and H3-receptor antagonistic properties⁹.

Histamine, impromidine and all its analogues contain a 4-(ω-aminoalkyl)imidazole fragment. The imidazole moiety is supposed to trigger the histamine H2 receptor via a l,3-prototropic shift¹⁰ (Fig. 4.2) involving one proton from a proton donating group at the histamine H2 receptor and the N^tele proton of the imidazole nucleus. Amino acids Asp-186 and Tyr-182 in transmembrane domain 5 are considered to be involved in this process (see receptor models in Chapter 4.1). The mechanism for dimaprit is less clear.

N N

NH₃⁺

B H H A N

N

NH₃⁺

H

B H A

τ π

Fig. 4.2. Interaction of the histamine monocation with the H2 receptor (1,3-prototropic shift) The structure-activity relationship studies on the H2 receptor agonism of dimaprit have revealed that the dimethylamino group has the same function as the amino group of histamine, while the isothiourea group of dimaprit and the imidazole group of histamine are regarded as bioisosteric groups³.

N N

S H

H H

N H

Site II H

Site III Site I

Fig. 4.3a. Interaction of dimaprit with the histamine H₂ receptor (S-fit)

N H

Site II Site III

Site I

S N H N

H H

H

Fig. 4.3b. Interaction of dimaprit with the histamine H₂ receptor (N-fit)

Green et al.¹¹ suggested the formation of a hydrogen bond between a proton of the histamine H2

receptor and the sulphur atom of dimaprit in the so-called S-fit (Fig. 4.3a) and the formation of a second hydrogen bond between a proton of one of the nitrogen atoms of the isothiourea group and another site of the same receptor. As with histamine two protons are involved, one from the receptor and one from the agonist, however, in contrast to histamine a tautomeric proton shift is impossible in the same region of the active site of the receptor. Durant et al³ considered a second possibility for the interaction of dimaprit with the histamine H2 receptor, so-called N-fit, placing the two nitrogen atoms of the isothiourea moiety of dimaprit in the same position as the two imidazole nitrogen atoms of histamine. As can be seen in Fig. 4.3b, this mechanism supposes an interaction between the receptor proton (site III) and a nitrogen atom of the isothiourea group and a proton of the second nitrogen atom of this same group and site II of the receptor, thus leaving the possibility for a 1,3-prototropic shift as proposed for the mechanism of histamine. However, quantum chemical calculations suggest that the S-fit, which includes the interaction of the NH-group and the S-atom of the isothiourea moity with the H2 receptor, is favourable. Donné-Op den Kelder et al.¹² performed fit procedures on both the N- and S-fit of dimaprit, showing that the conformation of the N-fit is not likely to be the "active" conformation.

On the basis of that, Impicciatore et al.¹³ first recognized the possibility of H2R agonistic activity of amino-5-(aminoethyl)thiazole, being a rigid analogue of dimaprit. Several substituted 2-aminothiazoles have indeed been shown to be active at the histamine H2 receptor; amthamine^14,15 3 (Fig 4.1) is the most potent H2 agonist in this series. These compounds show moderate to strong H2 receptor agonistic activity compared to histamine. Moreover, these thiazole analogues of histamine have very less effects on H1 and H3 receptors.

Realizing that 4-methyl-2-aminothiazoles are capable of stimulating the H2 receptor, a series of impromidine and arpromidine analogues, such as 7a¹⁶, was synthesized by replacing the 3-[(1H-imidazolyl-4-yl)propyl] group with a 3-(4-methyl-2-aminothiazol-5-yl)propyl moiety. Such compounds are reported to be selective agonists for the H2 receptor, in particular versus the H3

receptor¹⁷, i. e. the imidazoles and the aminothiazoles may be considered bioisosteres at histamine H2 but not at H3 receptors.

The strongly basic guanidino group (pKa value about 13), which is supposed to interact with Asp-98 in transmembrane domain 3, is essential for the agonistic activity of impromidine and analogues, but it is also the main reason for very low oral bioavailability, non-H2R-mediated effects and lack of penetration across the blood-brain barrier¹⁸. A major advancement was achieved by replacing the guanidino group by an acylguanidine moiety (see Chapter 3) resulting in potent H2R agonists with reduced basicity and improved pharmacokinetic properties.

Therefore, we decided to synthesise a series of N-acyl-N’-[3-(2-amino-4-methylthiazol-5-yl)propyl]guanidines. According to this concept the title compounds should combine advantages of the acylguanidines with improved H2R selectivity, in particular the histamine H3 receptor antagonistic activity was expected to be considerably reduced.

4.2. Chemistry

The 2-[3-(2-amino-4-methylthiazol-5-yl)propyl]isoindoline-1,3-dione (97) (Scheme 4.1) was synthesized according to Eriks^19-22 from the chloroketone 94 via phthalimide protection, regioselective bromination, and ring closure of 3-bromo-6-(phthalimido)hexan-2-one (96) with thiourea as presented in Scheme 1. Subsequently, the 2-amino group was Boc-protected and, finally, the phthalimide was deprotected by hydroginolysis.

H₃C Cl

Scheme 4.1: Synthesis of Boc-protected homoamthamine

The guanidino group was attached to the propyl chain by treating the amine 99 with di-Cbz-triflate-protected guanidine (23, Chapter 3, Scheme 3.5), followed by deprotection of the Cbz-groups by hydrogenolysis (Scheme 4.2).

Reagents: (i) 23, Et₃N, CHCl₃, rt, 93 %; (ii) H₂ / Pd-C, MeOH : THF (1:1), rt, 4 h

Scheme 4.2: Introduction of the guanidino group

The acylation of compound 101´ was very interesting. Under similar reaction condition as

The acylation of compound 101´ was very interesting. Under similar reaction condition as

Im Dokument Arpromidine-Related Acylguanidines: Synthesis and Structure- Activity Relationships of a New Class of Guanidine-Type Histamine H (Seite 108-0)